Formycin A and its N-methyl analogues, specific inhibitors of E-coli purine nucleoside phosphorylase (PNP): induced tautomeric shifts on binding to enzyme, and enzyme -> ligand fluorescence resonance energy transfer

Steady-state and time-resolved emission spectroscopy were used to study the interaction of Escherichia call purine nucleoside phosphorylase (PNP) with its specific inhibitors, viz. formycin B (FB), and formycin A (FA) and its N-methylated analogues, N(1)-methylformycin A (m(1)FA), N(2)-methylformyci n A (m(2)FA) and N(6)-methylformycin A (m(6)FA), in the absence and presenc e of phosphate (P-i). Complex formation led to marked quenching of enzyme t yrosine intrinsic fluorescence, with concomitant increases in fluorescence of FA and m(6)FA, independently of the presence of P-i. Fluorescence of m(1 )FA in the complex increased only in the presence of P-i, while the weak fl uorescence of FB appeared unaffected, independently of P-i. Analysis of the emission, excitation and absorption spectra of enzyme-ligand mixtures poin ted to fluorescence resonance energy transfer (FRET) from protein tyrosine residue(s) to FA and m(6)FA base moieties, as a major mechanism of protein fluorescence quenching. With the non-inhibitor m(2)FA, fluorescence emissio n and excitation spectra were purely additive. Effects of enzyme-FA, or enz yme-m(6)FA, interactions on nucleoside excitation and emission spectra reve aled shifts in tautomeric equilibria of the bound ligands. With FA, which e xists predominantly as the N(1)-H tautomer in solution, the proton N(1)-H i s shifted to N(2), independently of the presence of P-i. Complex formation with m(6)FA in the absence of P-i led to a shift of the amino-imino equilib rium in favor of the imino species, and increased fluorescence at 350 nm; b y contrast, in the presence of P-i, the equilibrium was shifted in favor of the amino species, accompanied by higher fluorescence at 430 nm, and a hig her affinity for the enzyme, with a dissociation constant K-d = 0.5 +/- 0.1 mu M, two orders of magnitude lower than that for m(6)FA in the absence of P-i (K-d = 46 +/- 5 mu M). The latter was confirmed by analysis of quenchi ng of enzyme fluorescence according to a modified Stern-Volmer model. Fract ional accessibility values (f(a)) varied from 0.31 for m(1)FA to 0.70 for F A, with negative cooperative binding of m(1)FA and FB, and non-cooperative binding of FA and m(6)FA. For all nucleoside ligands, the best model descri bing binding stoichiometry was one ligand per native enzyme hexamer. Fluore scence decays of PNP, FA and their mixtures were best fitted to a sum of tw o exponential terms, with average lifetimes [[tau]] affected by their inter actions. Complex formation resulted in a 2-fold increase in (tau) of FA, an d a 2-fold decrease in [tau] of enzyme fluorescence. The amplitude of the l ong-lifetime component also increased, confirming the shift of the tautomer ic equilibrium in favor of the N(2)-H species. The findings have been exami ned in relation to enzyme-nucleoside binding deduced from structural studie s. (C) 2000 Elsevier Science B.V. All rights reserved.